Visualisation of the mechanosensitive channel of large conductance in bacteria using confocal microscopy

Eur Biophys J. 2005 Jul;34(5):396-402. doi: 10.1007/s00249-005-0475-y. Epub 2005 Apr 6.

Abstract

The mechanosensitive channel of large conductance (MscL) plays an important role in the survival of bacterial cells to hypo-osmotic shock. This channel has been extensively studied and its sequence, structure and electrophysiological characteristics are well known. Here we present a method to visualise MscL in living bacteria using confocal microscopy. By creating a gene fusion between mscl and the gene encoding the green fluorescent protein (GFP) we were able to express the fusion protein MscL-GFP in bacteria. We show that MscL-GFP is present in the cytoplasmic membrane and forms functional channels. These channels have the same characteristics as wild-type MscL, except that they require more pressure to open. This method could prove an interesting, non-invasive, tool to study the localisation and the regulation of expression of MscL in bacteria.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Physiological Phenomena
  • Bacterial Proteins / chemistry
  • Biophysics / instrumentation
  • Biophysics / methods*
  • Cell Membrane / metabolism
  • Cell Proliferation
  • Cytoplasm / metabolism
  • Electrophysiology
  • Escherichia coli / metabolism
  • Escherichia coli Proteins / chemistry*
  • Green Fluorescent Proteins / chemistry
  • Green Fluorescent Proteins / metabolism
  • Ion Channels / chemistry*
  • Microscopy, Confocal / methods*
  • Plasmids / metabolism
  • Pressure
  • Recombinant Fusion Proteins / chemistry
  • Spheroplasts / metabolism
  • Time Factors

Substances

  • Bacterial Proteins
  • Escherichia coli Proteins
  • Ion Channels
  • MscL protein, E coli
  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins